Stress impacts multiple phases of opioid addiction and is associated with greater vulnerability to initiation of drug taking, more rapid transition from drug use to abuse, and higher rates of drug relapse, though the neural circuitry and mechanisms involved are largely unknown. A growing literature indicates the involvement of the dorsal raphe nucleus (DRN)-serotonin (5-hydroxytryptamine; 5-HT) system in some of the affective components of drug addiction that motivate drug taking and relapse. Acute opioids stimulate 5-HT neurotransmission via GABAergic disinhibition, potentially contributing a positive affective component to the motivation for drug seeking early in opioid addiction. By contrast, later in addiction, the 5-HT DRN system may also contribute to drug-seeking motivated by negative affect via its responsiveness to stressors and its interaction with the corticotropin-releasing factor (CRF) system. Our laboratory and others have shown that CRF-R1 receptors inhibit 5-HT DRN neurons via GABA interneurons. Preliminary data indicate a novel neuroadaptation within DRN-CRF circuits that is associated with vulnerability to stress-induced opioid relapse. Rats expressing extinction of either heroin intravenous self-administration (IVSA) or morphine conditioned place-preference (CPP) that are exposed to stress reinstate their previously extinguished heroin-seeking behavior or morphine CPP, an effect accompanied by sensitization of GABAA receptors on 5-HT DRN neurons. This neuroadaptation would render 5-HT DRN neurons vulnerable to inhibition by CRF-R1-GABA inputs. From these collective data, we hypothesize a dual role for the 5-HT system in opioid addiction: 1) early opioid exposure stimulates 5-HT DRN neurons, creating a positive affective state that contributes to opioid reward; 2) stress exposure in subjects with an opioid history sensitizes GABAA receptors on 5-HT DRN neurons, making them vulnerable to inhibition by CRF-R1 located on GABA afferents. The resulting 5-HT hypofunction creates a negative affective state that motivates opioid reinstatement. In specific aim 1, we will use electrophysiology to measure the dynamic changes in membrane properties and excitability of 5-HT DRN neurons ex vivo as they track with changes in affect and behavior over the course of a rat model of heroin IVSA, extinction and stress- induced reinstatement. In specific aim 2 we will use viral delivery of Designer Receptor Exclusively Activated by Designer Drugs in Tph2-Cre rats to examine the impact of manipulating 5-HT DRN neuronal activity in vivo in the behavioral model. In specific aim 3 we will use viral delivery of DREADDs in Crh-Cre or GAD-Cre rats to test the causal relationship between amygdala CRF afferents to the DRN and DRN GABA interneurons in the behavioral model. This proposal employs multiple ex vivo and in vivo approaches to dissect the role of 5-HT- GABA-CRF circuitry in a model of opioid addiction and relapse with the ultimate goal of identifying novel therapeutic targets to treat opioid addiction and prevent relapse.